Long-term storage, transport and archiving of nucleic acids on filter paper or chemically modified matrices is a well-known technique for preserving genetic material before the DNA or RNA is extracted and isolated in a form for use in genetic analysis such as the polymerase chain reaction (PCR). Thus, EP1563091 (Smith et al., Whatman) relates to methods for storing nucleic acids from samples such as cells or cell lysates. The nucleic acid is isolated and stored for extended periods of time, at room temperature and humidity, on a wide variety of filters and other types of solid support or solid phase media. Moreover, the document describes methods for storing nucleic acid-containing samples on a wide range of solid support matrices in tubes, columns, or multiwell plates.
WO/9003959 (Burgoyne) describes a cellulose-based solid support for the storage of DNA, including blood DNA, comprising a solid matrix having a compound or composition which protects against degradation of DNA incorporated into or absorbed on the matrix. This document also discloses methods for storage of DNA using the solid medium, and for recovery of or in situ use of DNA.
U.S. Pat. No. 5,705,345 (Lundin et al.) describes a method of nucleic acid preparation whereby the sample containing cells is lysed to release nucleic acid and the sample is treated with cyclodextrin to neutralize the extractant. The advantage of this method is that it eliminates the need for a separation step which is required for the removal of the lysis reagent.
GB2346370 (Cambridge Molecular Technologies Ltd) discloses a method involving applying a cellular sample containing nucleic acid to a filter, lysing the cells, then retaining the nucleic acid on the filter while removing contaminants.
WO9618731 (Deggerdal) describes a method of isolating nucleic acid whereby the sample is bound to a solid support and the sample is contacted with a detergent and subsequent steps performed to isolate the nucleic acid.
WO0053807 (Smith) discloses a medium for the storage and lysis of samples containing genetic material which can be eluted and analysed. The medium is coated with a lysis reagent and optionally with a weak base, a chelating agent, a surfactant or uric acid.
Ambient storage of biological samples is being seen as a better alternative to storage at low temperature. FTA™ (GE Healthcare) can be used for the storage of small sample sizes of approximately 100-200 ng of DNA. However there is a growing need for storage of larger sample volumes at ambient temperatures. A number of international prospective studies are recruiting many thousands of participants in an effort to investigate links between the living environment, life-style and genetics with the onset of disease. For example the EPIC prospective study on diet and cancer and the Canadian Partnership for Tomorrow Project. The studies rely on in-depth analysis of the participants at the outset and subsequent analysis of DNA following diagnosis of a significant life-threatening disease sometime in the future. Moderate blood volumes of 4 ml or more are required when conducting large cohort studies to allow genetic analysis of samples using contemporary and evolving techniques. Ideally samples should contain material that would allow additional investigation of other important molecules, for example long non-coding RNA. Therefore there is a need for a means to store large volumes of cell nuclei, which contains DNA, RNA and numerous other proteins, at ambient temperatures for current and future analytical studies.
Previous nuclei capture devices have included Nuclitip (GE Healthcare; described in U.S. Pat. No. 5,447,864, Kenrick et al.). The device consists of a microfilament weave of the tip of a pipette that processes up to 10 ml of fresh blood. The blood undergoes controlled lysis; the cell membrane is lysed leaving the majority of nuclei membrane intact. A planar treated membrane is located on the exterior of the Nuclitip pipette tip completely covering the tip's aperture such that the sample is filtered before entry into the tip and any DNA and nuclei present in the sample binds to the filter. The pipette tip is then washed with phosphate buffered saline (PBS) to remove any contaminants. U.S. Pat. No. 5,447,864 describes the method of separating cell components of cells using the nuclitip method and the possibility of storing the nuclei for longer periods on the membrane at −20° C. or below and for short periods if kept at 4° C. However the document does not disclose or suggest the long term storage of cell nuclei at ambient temperatures. In addition the document suggests the use of standard nucleic acid extraction techniques including the use of detergents to lyse the nuclear membrane.
There is therefore a need for an improved and simplified process for capturing and storing large quantities of nucleic acid, including DNA and RNA, at ambient temperature. The present invention addresses this problem and provides methods and kits which can be used for single step storage and extraction of nucleic acid from solid supports, particularly cellulose-derived supports.